Crystal structures of halohydrin hydrogen‐halide‐lyases from Corynebacterium sp. N‐1074

Halohydrin hydrogen‐halide‐lyase (H‐Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2‐diol. Until now, six different H‐Lyases have been studied. These H‐Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N‐1074 has two different isozymes of H‐Lyase, HheA (A‐type) and HheB (B‐type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H‐Lyases. Among the B‐type H‐Lyases, HheB shows relatively high enantioselectivity compared to that of HheB_GP1. This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3‐dicyano‐2‐propanol at the catalytic site in the crystal structure of the HheB‐DiCN complex suggests that the product should be (R)‐epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity. Proteins 2015; 83:2230–2239. © 2015 Wiley Periodicals, Inc.     

A New sp2‐sp2 Dialkylethylene‐Bridged Heptacyclic Ladder‐Type Arene for High Efficiency Polymer Solar Cells

A new dithienobenzo‐carbazole (DTBC) heptacyclic arene using sp²‐sp² dialkylethylene moiety as the bridge was successfully synthesized by Suzuki‐Miyaura cross‐coupling reaction. This facile benzoannulation planarizes the conjugated framework with the concomitant introduction of four octyl substituents, making the newly designed copolymer PDTBCDTBT exhibit solution processability, superior absorption ability, lower‐lying HOMO energy level, and crystalline nature. The solar cell using PDTBCDTBT has produced a high efficiency of 6.2%, which outperforms the corresponding sp³‐hybridized C‐bridged, Si‐bridged and N‐bridged analogous polymers.     

New Pyran‐2‐ones from Alkalophilic Actinomycete,Nocardiopsis alkaliphila sp. Nov. YIM‐80379

Two new pyran‐2‐ones, nocardiopyrones A and B ( 1 and 2 , resp.), along with four known compounds, pyridinols 3 – 5 , and 1‐acetyl‐β‐carboline ( 6 ) were isolated from the alkalophilic actinomycete Nocardiopsis alkaliphila sp. nov. YIM‐80379. Their structures were established on the basis of spectroscopic analysis, CD spectra, and the quantum‐chemical ECD calculation. Pyridinols 3 – 5 were isolated from a natural source for the first time. Compounds 1 and 2 showed weak antibacterial activities against Pseudomonas aeruginosa, Enterobacter aerogenes, and Escherichia coli with MIC values of 20–48 μM . Compound 2 showed weak antimicrobial activities against Candida albicans and Staphylococcus aureus with MIC values of 24 and 48 μM , respectively.     

Low‐Overpotential Electrocatalytic Water Splitting with Noble‐Metal‐Free Nanoparticles Supported in a sp3 N‐Rich Flexible COF

Covalent organic frameworks (COFs) are crystalline organic polymers with tunable structures. Here, a COF is prepared using building units with highly flexible tetrahedral sp³ nitrogens. This flexibility gives rise to structural changes which generate mesopores capable of confining very small (<2 nm sized) non‐noble‐metal‐based nanoparticles (NPs). This nanocomposite shows exceptional activity toward the oxygen‐evolution reaction from alkaline water with an overpotential of 258 mV at a current density of 10 mA cm^?2. The overpotential observed in the COF‐nanoparticle system is the best in class, and is close to the current record of ≈200 mV for any noble‐metal‐free electrocatalytic water splitting system—the Fe–Co–Ni metal‐oxide‐film system. Also, it possesses outstanding kinetics (Tafel slope of 38.9 mV dec^?1) for the reaction. The COF is able to stabilize such small‐sized NP in the absence of any capping agent because of the COF–Ni(OH)₂ interactions arising from the N‐rich backbone of the COF. Density‐functional‐theory modeling of the interaction between the hexagonal Ni(OH)₂ nanosheets and the COF shows that in the most favorable configuration the Ni(OH)₂ nanosheets are sandwiched between the sp³ nitrogens of the adjacent COF layers and this can be crucial to maximizing their synergistic interactions.     

Production of Host‐selective SV‐toxins by Stemphylium sp. Causing Brown Spot of European Pear in Japan

Recently, a new disease known as ‘brown spot of European pear’ caused by Stemphylium sp. appeared on the leaves, twigs and fruits of the cultivar Le Lectier in Niigata Prefecture, Japan. Because Stemphylium vesicarium (teleomorph: Pleospora allii), which causes a similar disease in Europe, has been shown to produce host‐selective SV‐toxins in culture filtrates (CFs), SV‐toxin production by Stemphylium sp. in Japan was investigated. In pathogenicity tests, the pathogen induced severe necrotic spots on the leaves of the European pear cultivar Le Lectier, slight spots on cultivar La France and slight or no spots on cultivar Bartlett. The Japanese pear cultivar Nijisseiki was not affected by the pathogen. Culture filtrates of the pathogen were tested for phytotoxicity on cultivars by a leaf necrosis assay. The sensitivity of cultivars to the CFs was consistent with the susceptibility of cultivars to the pathogen infection, indicating the presence of host‐selective toxins. The toxins were purified from the CFs according to the procedure reported for SV‐toxin purification in S. vesicarium. The results indicated that Stemphylium sp. in Japan produces the same SV‐toxins as S. vesicarium in Europe.     

Molecular architecture of KedS8, a sugar N‐methyltransferase from Streptoalloteichus sp. ATCC 53650

Kedarcidin, produced by Streptoalloteichus sp. ATCC 53650, is a fascinating chromoprotein of 114 amino acid residues that displays both antibiotic and anticancer activity. The chromophore responsible for its chemotherapeutic activity is an ansa‐bridged enediyne with two attached sugars, l ‐mycarose, and l ‐kedarosamine. The biosynthesis of l ‐kedarosamine, a highly unusual trideoxysugar, is beginning to be revealed through bioinformatics approaches. One of the enzymes putatively involved in the production of this carbohydrate is referred to as KedS8. It has been proposed that KedS8 is an N‐methyltransferase that utilizes S‐adenosylmethionine as the methyl donor and a dTDP‐linked C‐4′ amino sugar as the substrate. Here we describe the three‐dimensional architecture of KedS8 in complex with S‐adenosylhomocysteine. The structure was solved to 2.0 Å resolution and refined to an overall R‐factor of 17.1%. Unlike that observed for other sugar N‐methyltransferases, KedS8 adopts a novel tetrameric quaternary structure due to the swapping of β‐strands at the N‐termini of its subunits. The structure presented here represents the first example of an N‐methyltransferase that functions on C‐4′ rather than C‐3′ amino sugars.     

Anabaena sp. DyP‐type peroxidase is a tetramer consisting of two asymmetric dimers

DyP‐type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure‐based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49–55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP‐type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224‐Cys224 disulfide‐linked dimers. The tetramer of wild‐type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration‐dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non‐crystallographic, 2‐fold symmetry in the asymmetric unit, two subunits forming the Cys224‐Cys224 disulfide‐linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure‐based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes. Proteins 2016; 84:31–42. © 2015 Wiley Periodicals, Inc.     

Single‐cell sequencing unveils the lifestyle and CRISPR‐based population history of Hydrotalea sp. in acid mine drainage

Acid mine drainage (AMD) is characterized by an acid and metal‐rich run‐off that originates from mining systems. Despite having been studied for many decades, much remains unknown about the microbial community dynamics in AMD sites, especially during their early development, when the acidity is moderate. Here, we describe draft genome assemblies from single cells retrieved from an early‐stage AMD sample. These cells belong to the genus Hydrotalea and are closely related to Hydrotalea flava. The phylogeny and average nucleotide identity analysis suggest that all single amplified genomes (SAGs) form two clades that may represent different strains. These cells have the genomic potential for denitrification, copper and other metal resistance. Two coexisting CRISPR‐Cas loci were recovered across SAGs, and we observed heterogeneity in the population with regard to the spacer sequences, together with the loss of trailer‐end spacers. Our results suggest that the genomes of Hydrotalea sp. strains studied here are adjusting to a quickly changing selective pressure at the microhabitat scale, and an important form of this selective pressure is infection by foreign DNA.     

The Chemical Constituents of Endophytic Fungus Trichoderma sp. MFF‐1

A fungal strain named MFF‐1 was isolated from the flower of Pyrethrum cinerariifolium. Based on the sequence at the internal transcribed spacer (ITS) region, this strain was identified as a Trichoderma sp. Two new compounds, including a mitorubrin derivative and its potential biogenetic precursor, together with a known compound, were isolated from the cultures of the endophytic fungus. Their structures were established by spectroscopic methods and determined to be (3S*,6R*,7R*)‐3,4,5,6,7,8‐hexahydro‐7‐hydroxy‐7‐methyl‐8‐oxo‐3‐[(E)‐prop‐1‐enyl]‐1H‐isochromen‐6‐yl 2,4‐dihydroxy‐6‐methylbenzoate ( 1 ), named deacetylisowortmin, (E)‐2‐(hydroxymethyl)‐3‐(2‐hydroxypent‐3‐enyl)phenol ( 2 ), and wortmannin ( 3 ). All compounds were assayed for antimicrobial activity. Compound 3 showed activity against Candida albicans and Bacillus cereus.     

Enhanced lipid production in Nannochloropsis sp. using fluorescence‐activated cell sorting

To advance the utilization of microalgae as a viable feedstock for biodiesel production, the intracellular lipid content of three strains of the marine microalgae Nannochloropsis sp. was enhanced using flow cytometry (FC) coupled with cell sorting. Total lipid content was doubled to 55% (biomass dry weight) in the sorted, daughter cells of Nannochloropsis (strain 47) after consecutive three rounds of cell sorting, and this trait was maintained for approximately 100 subsequent cell generations. In addition, daughter cells had a fatty acid profile similar to that of the parent, wild‐type strain. The study demonstrates that FC coupled with cell sorting is a powerful tool for the enhancement of intracellular lipid content in microalgae exploited for biodiesel feedstock.     

Homo‐C‐nucleoside analogs IV. Synthesis of 4‐(2,5‐anhydro‐D‐altro‐pentitol‐1‐yl)‐2‐phenyl‐2H‐1,2,3‐triazole homo‐C‐nucleoside. CD assignment of the absolute configuration of the carbon bridge

Dehydrative cyclization of 4‐(D‐altro ‐pentitol‐1‐yl)2‐phenyl‐2H ‐1,2,3‐triazole in basic medium with one moler equivalent of p‐toluene sulfonyl chloride in pyridine solution gave the homo‐C‐ nucleoside 4‐(2,5‐anhydro‐D‐altro ‐1‐yl)‐2‐phenyl‐2H ‐1,2,3‐triazole. The structure and anomeric configuration was determined by acylation, nuclear magnetic resonance (NMR), and mass spectroscopy. The stereochemistry at the carbon bridge of homo‐C‐ nucleoside 2‐phenyl‐2H ‐1,2,3‐triazoles was determined by circular dichroism (CD) spectroscopy.     

Functional redundancy between flavodiiron proteins and NDH‐1 in Synechocystis sp. PCC 6803

In oxygenic photosynthetic organisms, excluding angiosperms, flavodiiron proteins (FDPs) catalyze light‐dependent reduction of O₂ to H₂O. This alleviates electron pressure on the photosynthetic apparatus and protects it from photodamage. In Synechocystis sp. PCC 6803, four FDP isoforms function as hetero‐oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase‐like complex (NDH‐1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH‐1 types have been characterized in cyanobacteria: NDH‐1₁ and NDH‐1₂, which function in respiration; and NDH‐1₃ and NDH‐1₄, which function in CO₂ uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants (?flv1 and Δflv3) and the double NDH‐1 mutants (?d1d2, which is deficient in NDH‐1_1,2 and ?d3d4, which is deficient in NDH‐1_3,4), we studied triple mutants lacking one of Flv1 or Flv3, and NDH‐1_1,2 or NDH‐1_3,4. We show that the presence of either Flv1/3 or NDH‐1_1,2, but not NDH‐1_3,4, is indispensable for survival during changes in growth conditions from high CO₂/moderate light to low CO₂/high light. Our results show functional redundancy between FDPs and NDH‐1_1,2 under the studied conditions. We suggest that ferredoxin probably functions as a primary electron donor to both Flv1/3 and NDH‐1_1,2, allowing their functions to be dynamically coordinated for efficient oxidation of photosystem I and for photoprotection under variable CO₂ and light availability.